Human anti-epidermal growth factor receptor single-chain antibodies

ABSTRACT

Human anti-epidermal growth factor receptor (EGFR) single-chain antibodies (scFvs) were isolated from a human IgM phage display library using purified epidermal growth factor receptor as antigen. Two isolates with different amino acid sequences were identified by ELISA as epidermal growth factor receptor-specific. The scFvs bind to the full length epidermal growth factor receptor and the truncated and/or mutated epidermal growth factor receptor on human cells. These anti-EGFR-scFvs are useful as therapeutic and/or diagnostic agents.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims benefit of patent application U.S.Serial No. 60/240,353, filed Oct. 13, 2000, now abandoned.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields ofmolecular biology and monoclonal antibody technology. More specifically,the present invention relates to human single-chain antibodies that bindspecifically to the epidermal growth factor receptor.

[0004] 2. Description of the Related Art

[0005] The epidermal growth factor receptor (EGFR) is a 170 kDatransmembrane glycoprotein consisting of an extracellular ligand bindingdomain, a transmembrane region and an intracellular domain with tyrosinekinase activity. The binding of growth factors, EGF or TGFα, to theepidermal growth factor receptor results in receptor dimerization,auto-phosphorylation and induction of a tyrosine kinase cascade, leadingultimately to DNA synthesis and cell division.

[0006] The epidermal growth factor receptor gene (c-erb-1), located onchromosome 7, is homologous to the avian erythroblastosis virus oncogene(v-erbB) that induces malignancies in chickens. The v-erbB gene encodesfor a truncated protein product that lacks the extracellular ligandbinding domain. The tyrosine kinase domain of the epidermal growthfactor receptor has been found to have 97% homology to the v-erbBtransforming protein.

[0007] The epidermal growth factor receptor is overexpressed in a numberof malignant human tissues when compared to their normal tissuecounterparts. The gene for the receptor is both amplified andoverexpressed in a number of cancer cells. Overexpression of theepidermal growth factor receptor is often accompanied by theco-expression of the growth factors, EGF and TGFα, suggesting that anautocrine pathway for control of growth may play a major part in theprogression of tumors.

[0008] A high incidence of overexpression, amplification, deletion andstructural rearrangement of the gene coding for the epidermal growthfactor receptor has been found in biopsies of brain tumors. In fact, theamplification of the epidermal growth factor receptor gene inglioblastoma multiforme tumors is one of the most consistent geneticalterations known, with the EGFR being overexpressed in approximately40% of malignant gliomas. In addition to glioblastomas, abnormalepidermal—growth factor receptor expression has also been reported in anumber of squamous epidermoid cancers and breast cancers. Many patientswith tumors that overexpress the epidermal growth factor receptor have apoorer prognosis than those who do not. Consequently, therapeuticstrategies which can potentially inhibit or reduce the aberrantexpression of the EGFR are of great interest as potential anti-canceragents.

[0009] Since the advent of hybridoma technology to produce murinemonoclonal antibodies (mAbs) developed by Milstein and Köhler in 1975(1), the therapeutic potential of antibodies is beginning to come tofruition for cancer therapy. There are many reports describing a fewantibodies which inhibit cell proliferation of epidermal growth factorreceptor-overexpressing cell lines (2-6). One such mouse antibody, mAb225, was shown to inhibit cell proliferation and block ligand-inducedepidermal growth factor receptor tyrosine kinase activity (2-3, 7).Further analysis showed mAb 225 induced a G₁ growth arrest and activatedan apoptotic pathway after a 24 h exposure to increasing concentrationsof antibody (8).

[0010] Other monoclonal antibodies which bind to the epidermal growthfactor receptor and block ligand binding also show promise for cancertherapy. One group of rat monoclonal antibodies showed a dramaticantitumor effect in xenograft mouse models, with one antibody, ICR62curing 4 out of 8 mice of the tumor (9). However, the problem with ratand mouse monoclonal antibodies or even the human-mouse chimericantibody is the possibility of an immune or allergic response withprolonged treatment (10-13).

[0011] In order to avoid the human anti-murine antibody (HAMA) responsein humans due to the repeated administration of murine mAbs, it ispreferable to use human antibody in therapy or diagnostics. A 100% humanmonoclonal antibody against the epidermal growth factor receptor,E7.6.3, has been shown to completely eradicate human tumor xenografts inmice (4). This antibody is expected to elicit a minimal immune responsein humans and shows promise for future cancer therapy. However due tothe heterologous vascular structure around the tumor and the molecularsize of the antibodies, monoclonal antibodies penetrate the tumor poorlyand are unevenly distributed around the tumor.

[0012] In order to improve on the use of monoclonal antibodies, intactmonoclonal antibodies have been reduced in size to antibody fragments orsingle-chain antibodies (scFvs). Therefore the development of humananti-EGFR scFvs will enhance its use as a diagnostic and/or therapeuticagent. One advantage of single-chain antibodies is their ability topenetrate deeper into the tumor (14). Thus, these molecules maypotentially be more efficacious than intact antibodies for systemicadministration. Also single-chain antibodies can be expressedintracellularly (intrabodies) and targeted to a subcellular compartmentof the tumor cell or be secreted by the tumor cell and bind in anautocrine/paracrine fashion.

[0013] The prior art is deficient in the lack of a 100% humansingle-chain antibody that binds to the epidermal growth factorreceptor. The present invention fulfills this longstanding need anddesire in the art.

SUMMARY OF THE INVENTION

[0014] The present invention provides a 100% human single-chain antibody(scFv) which binds to the epidermal growth factor receptor. Twosingle-chain antibodies were isolated from a human IgM phage displaylibrary using purified epidermal growth factor receptor as antigen, andidentified by ELISA as epidermal growth factor receptor-specific.Sequence analysis confirmed the two isolates as individual clones basedon differences in their -nucleotide and putative amino acid sequences.One single-chain antibody was shown to bind to the native full lengthepidermal growth factor receptor and the truncated and/or mutatedepidermal growth factor receptor on human cells.

[0015] The present invention is directed to a human anti-epidermalgrowth factor receptor single-chain antibody having a sequence of SEQ IDNo. 1 (clone 6) or SEQ ID No. 2 (clone 63), as well as DNA molecules andexpression vectors that encode for the expression of the claimed humananti-epidermal growth factor receptor single-chain antibody.

[0016] The present invention is also drawn to a pharmaceuticalcomposition comprising the disclosed human anti-epidermal growth factorreceptor scFv and a therapeutic and/or diagnostic agent. Preferably, thetherapeutic and/or diagnostic agent can be a toxin, a chemotherapeuticagent, a radioisotope, a transition metal or a gene therapy vector.

[0017] The present invention is also drawn to a method of treating orimaging a tumor, comprising the step of administering to a patient inneed of such treatment or detection an effective amount of aradiolabeled anti-EGFR single-chain Fv of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0019]FIG. 1 shows the phagemid, pSEX81, which is optimized for surfaceexpression on the M13 bacteriophage. Depicted here is a single-chainantibody cloned into the multiple cloning site, in-frame with the pelBleader sequence and gene III M13 protein.

[0020]FIG. 2 shows the putative amino acid sequences of anti-EGFR scFvclones pSEX81-6 and pSEX81-63. Clones were sequenced both directionsusing primers from the pelB leader sequence, the gene III protein andtwo complementary primers annealing to the alpha tubulin linkersequence. The heavy chain variable region (V_(H)) and light-chainvariable region (V_(L)) are identified with their respective CDRs (bold)as described (16). *identifies identical amino acids.

[0021]FIG. 3 shows the eukaryotic secreting plasmid, pSECTAG/Bpu/neowhich was modified from pSECTAG/Friendly (Invitrogen). The neomycin genereplaced the zeomycin gene and a Bpu 11021 restriction enzyme site wasadded in-frame with the 1 g leader sequence.

[0022]FIG. 4 shows the analysis of secretory anti-EGFR-scFv from U87MGcell lines stably transfected with vector (pSECTAG) or anti-EGFR scFvclone 6 (p6.34). FIG. 4A shows immunoprecipitation of scFv from celllysates of pSECTAG (lane 1) and p6.34 (lane 2) using anti-myc antibody.FIG. 4B shows immunoprecipitation of secretory scFv from culture mediumof pSECTAG (lane 1) and p6.34 (lane 2) using anti-alpha tubulinantibody. Anti-myc antibody was used to detect scFv for both immunoblotsin FIGS. 4A and 4B. FIG. 4C shows ELISA using cell culture medium, orculture medium from pSECTAG, p6.34 or anti-EGFR mAb (Sigma) to detectbinding to the epidermal growth factor receptor antigen. ScFvs weredetected using anti-myc-HRP antibody and anti-epidermal growth factorreceptor mAb was detected with anti-mouse IgG-HRP antibody and developedwith OPD (Sigma).

[0023]FIG. 5 shows FACS analysis for the detection of anti-epidermalgrowth factor receptor scFv bound to the extracellular domain of theepidermal growth factor receptor. The cells, U87MG, U87MG.wtEGFR andU87MG.AEGFR were incubated with culture medium, culture medium frompSECTAG or p6.34 for 30 min at 4° C. The cells were washed, thenincubated with rat anti-alpha tubulin followed by FITC-labeled anti-ratantibody.

[0024] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Because the administration of murine monoclonal antibodies tohumans resulted in human anti-murine antibody (HAMA) response, hinderingany therapeutic and/or diagnostic potential, the monoclonal antibodieshad to be modified. Genetic engineering techniques were used to develophuman-murine chimeric monoclonal antibodies. An alternative solution wasto develop single-chain antibodies (scFvs). Initially murine scFv wereisolated and now technology has progressed to screening naive humanphage display libraries for therapeutically and/or diagnostically usefulsingle-chain antibodies of human origin (30-32).

[0026] Over the past 10 years, a variety of mouse and human single-chainantibodies have been isolated, including mouse scFvs which bind to thecell surface receptors, epidermal growth factor receptor and erbB-2 (23,27, 33). Cell proliferation was inhibited when EGFR-overexpressing celllines were transfected with a plasmid encoding murine anti-EGFR scFvstargeted to the endoplasmic reticulum (ER) or secretory pathway (23,27). Inhibition of cell proliferation was obtained whenerbB-2-overexpressing cell lines were transfected with a plasmidresulting in the production of murine single-chain antibodies whichbinds the erbB-2 receptor in the ERbut no inhibition of cellproliferation was detected with a cytoplasm-targeted scFv (33). Eventhough both of the single-chain antibodies bound to the extracellulardomain of their respective receptor they were expressed as anintracellular scFv (intrabody). The intrabodies were directed to thelumen of the ER to bind the receptor as it was being processed forglycosylation, thereby decreasing the amount of receptor expressed onthe plasma membrane and inhibiting cell proliferation. To obtain thegreatest anti-proliferative effect, the optimal expression and targetingof anti-EGFR single-chain antibodies to the subcellular componentsinvolved in epidermal growth factor receptor expression should beundertaken.

[0027] The ability of a single-chain antibody to inhibit tumor cellproliferation has considerable potential for cancer gene therapy on itsown merit. Moreover, the ability of a scFv to sensitize tumor cells toradiation or chemotherapy treatments will enhance their therapeuticpotential. Tumor cells either transfected with scFv expressing plasmidDNA or transduced with viral vectors have shown an increased sensitivityto radiation and/or chemotherapy drugs in vitro and in vivo (34, 35).The anti-erbB-2 scFv, pGT21, was shown to sensitize ovarian cancercells, SKOV3, to cis-DDP (34). The increased sensitization to cis-DDPwas shown to be related to the down-modulation of the erbB-2 protein bytargeting the scFv to the ER. The same scFv also sensitized tumor cellsto radiation in vitro and in vivo (35). However, a major limitation forthese scFvs is the fact that the majority are murine and thereforepotentially immunogenic in human.

[0028] The present invention discloses a 100% human single-chainantibody (scFv) which binds to the epidermal growth factor receptor. TwoscFvs were isolated from a human IgM phage display library usingpurified epidermal growth factor receptor as antigen, and identified byELISA as epidermal growth factor receptor-specific. Sequence analysisconfirmed the two isolates as individual clones based on differences intheir nucleotide and putative amino acid sequences. One scFv was shownto bind to the native full length epidermal growth factor receptor andthe truncated and/or mutated epidermal growth factor receptor on humancells.

[0029] As used herein, the term “monoclonal antibody” means an antibodycomposition recognizing a discrete antigen determinant. It is notintended to be limited with regard to the source of the antibody or themanner in which it is made.

[0030] As used herein, single chain antibodies or scFvs are polypeptideswhich consist of the variable (V) region of an antibody heavy chainlinked to the V region of an antibody light chain with or without aninterconnecting linker. This comprises the entire antigen binding site,and is the minimal antigen binding site. These single-chain antibodiesmay be produced in bacteria, yeast or eukaryotic cells.

[0031] An “antigen-binding site” refers to the part of an immunoglobulinmolecule that participates in antigen binding. The antigen binding siteis formed by amino acid residues of the N-terminal variable regions ofthe heavy and light chains. Three highly divergent stretches within theV regions of the heavy and light chains are referred to as“hypervariable regions” which are interposed between more conservedflanking stretches known as “framework regions” or “FRs”. In an antibodymolecule, the three hypervariable regions of a light chain and the threehypervariable regions of a heavy chain are disposed relative to eachother in three dimensional space to form an antigen binding “surface”.This surface mediates recognition and binding of the target antigen. Thethree hypervariable regions of each of the heavy and light chains arereferred to as “complementarity determining regions” or “CDRs” and arecharacterized, for example by Kabat et al., Sequences of proteins ofimmunological interest, 4th ed., U.S. Dept. Health and Human Services,Public Health Services, Bethesda, Md. (1987).

[0032] As an agent by itself, the scFv disclosed herein may inhibitand/or block the growth of epidermal growth factor receptor-expressinghuman cells. The human anti-epidermal growth factor receptor scFv mayalso induce apoptosis and cell death in human cells that expressepidermal growth factor receptor. A toxin, chemotherapeutic agent, atransition metal or radioisotope generally known in the art can becovalently or non-convalently conjugated to the scFv of the presentinvention, which would then target the agent to epidermal growth factorreceptor-expressing human cells. The scFv disclosed herein may also beused as a part of a bi-specific scFv or some other combination witheither itself or another scFv. Furthermore, all or portions of the scFvdisclosed herein may be used to target viral or bacterial gene therapyvectors or other agents to bind to epidermal growth factorreceptor-expressing human cells. The portions of the scFv could be assmall as one complementarity determining region (CDR) or a combinationof CDRs from one or both variable regions.

[0033] One object of the present invention is to target a scFv to aparticular cellular process as a powerful therapeutic technique.Combining the targeted scFv with a gene-based therapeutic approach mayenhance the efficacy of single-chain antibodies. The realization of thegoals of the current invention will allow for the design of cancer genetherapy treatment using intratumoral injection of a viral vector forsuccessful transduction of a therapeutic scFv to be used in combinationwith radiation and/or chemotherapy drugs.

[0034] A number of methods can be used to deliver the single-chainantibodies of the present invention to tumor cells. In the ex vivomethod, the scFv is expressed in bacterial cells (14, 36-37) oreukaryotic cells (24), then isolated and purified prior toadministration to the tumor cell lines or tumors implanted in mice. Thepurified scFv may be administered directly or labeled with aradioisotope, transition metal or toxin prior to administration (38-41).Also, the single-chain antibodies can be engineered to express abacterial toxin protein on the C-terminal end to enhance the therapeuticpotential of the scFv (37). The administration of an ex vivo producedscFv will rapidly localize and penetrate the tumor before being quicklycleared from the circulatory system (14, 38-39).

[0035] The in vivo expression of single-chain antibodies can result fromtransiently or stably transfecting cells with DNA (24-25) or transducingcells with viral vectors. DNA transfer can be accomplished by a varietyof standard techniques, such as calcium phosphate, DEAE dextran,electroporation or lipophilic reagents or by using a viral vector totransport the DNA into the cells. Most DNA transfection methods workvery well for in vitro experiments; however, viral vectors may be moreadvantageous for in vivo protocols. Viral vectors commonly used for genetherapy include retrovirus, adenovirus, adeno-associated virus andherpesvirus.

[0036] The invention also includes biologically functional fragments ofthe single-chain antibodies described in this specification.Biologically functional fragments are those fragments sufficient forbinding of the antibody fragment to epidermal growth factor receptor.Functional fragments include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies of the present invention.“Substantially the same” amino acid sequence is defined herein as asequence with at least 70% percent homology to an amino acid sequence ofan antibody of the present invention.

[0037] Furthermore, other “substantially homologous” modified antibodypolypeptides can be readily designed and manufactured utilizing variousrecombinant DNA techniques known to those skilled in the art.Modification of the genes may be readily accomplished by a variety ofwell-known techniques such as site-directed or random mutagenesis. Thesemodifications can include amino acid additions, deletions,substitutions, preferably conservative, and other changes in thesequence of the polypeptide while retaining the appropriate property orbiological activity.

[0038] Alternatively, polypeptide fragments comprising only a portion ofthe primary antibody structure and possessing binding and/or effectoractivities may be produced. Also because, like many genes, theimmunoglobulin-related genes contain separate functional regions, eachhaving one or more distinct biological activities, the genes may befused to functional regions from other genes to produce fusion proteins(e.g. immunotoxins) having novel properties or novel combinations ofproperties.

[0039] The current invention is directed to a human anti-epidermalgrowth factor receptor single-chain antibody having a sequence of SEQ IDNo. 1 (clone 6) or SEQ ID. No. 2 (clone 63), as well as DNA moleculesand expression vectors that encode for the expression of the claimedhuman anti-epidermal growth factor receptor scFv.

[0040] The present invention is also drawn to a pharmaceuticalcomposition comprising the disclosed human anti-epidermal growth factorreceptor scFv and a therapeutic or diagnositic agent. Preferably, thetherapeutic or diagnositic agent can be a toxin, a chemotherapeuticagent, a transition metal, a radioisotope or a gene therapy vector.

[0041] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1

[0042] Isolating EGFR-Specific Human Single-Chain Antibody (scFv)

[0043] In the screening of the phage library for anti-epidermal growthfactor receptor scFv, an IgM scFv display library with a calculatedcomplexity of 2×10⁷ independent clones was constructed in pSEX81(FIG. 1) as described (15) using peripheral leukocyte cDNA prepared fromhealthy donors. The IgM phage display library was screened forexpression of scFvs which specifically bind the EGF receptor (EGFR). Thephage library was suspended in 500 ml 2xYT-GA medium (17 g Tryptone, 10g yeast extract, 100 mM glucose, 100 μg/ml ampicillin and H₂₀ to 1liter) to an initial OD₆₀₀ of 0.025. The cells were grown with shaking(240 rpm) at 37° C. until an OD₆₀₀ of 0.1 at which point the cells weresuperinfected with helper phage, M13K07 (Amersham Pharmacia Biotech), atan MOI of 20. After the addition of helper phage, the cells were mixedgently and left undisturbed for 15 min at 37° C. followed by shaking(240 rpm) for 45 min. The medium was replaced by separating the bacteriaat 1500×g for 10 min at room temperature (RT), then the -bacterialpellet was suspended in 500 ml 2xYT-AK medium (17 g Tryptone, 10 g yeastextract, 50 μg/ml kanamycin, 100 μg/ml ampicillin and H₂₀ to 1 liter).The culture was incubated at 37° C. with shaking (240 rpm) for 7 h. Thecells were separated from the medium by centrifugation at 6500×g for 15min at 4° C. The bacteriophage was precipitated out of the supernatantby adding ⅕ volume PEG/NaCl solution (200 g PEG-600, 146.1 g NaCl, up to1 liter with H₂O) and incubating the medium overnight at 4° C.

[0044] During the PEG precipitation, affinity purified EGFR (Sigma) in100 mM sodium carbonate, pH 9.6 was adsorbed to a MaxiSorb Immunotube(Nunc, Rochester, NY) at a concentration of 5-10 μg/ml for 18 h at 4° C.The next day, the immunotube was washed 3 times with PBS, then blockedwith 2% skim milk in PBS-T (PBS with 0.05% Tween-20) for 2-3 h at roomtemperature (the immunotubes were blocked with 0.5% casein-PBS for thesecond round and 2% skim milk-PBS for the third round). The immunotubewas washed 3 times with PBS-T, then stored at 4° C. until ready to use.

[0045] The PEG precipitated bacteriophage was separated from thesupernatant by high speed centrifugation (10,000×g for 20 min at 4° C.).The pellet was suspended in 4 ml ice-cold phage dilution buffer (10 mMTris-HCl, pH 7.5, 20 mM NaCl, 2 mM EDTA). The bacteriophage lysate wasclarified at 12,000×g for 5 min at 4° C. The supernatant was collectedand stored at 4° C. until the colony forming units (cfu) titer wasdetermined.

[0046] To determine the cfu titer, an aliquot of the PEG-concentratedbacteriophage was diluted 10-fold up to 10⁻¹⁰ dilution, then 10 μl ofthe 10⁻⁷ to 10⁻¹⁰ dilutions was added to 90 μl of an exponentiallygrowing XL1-Blue culture in LB-tet broth (10 g tryptone, 5 g yeastextract, 0.5 g NaCl, 15 μg/ml tetracycline in 1 liter H₂O). The viruswas allowed to adsorb for 20-30 min at RT, then mixed with 3 ml ofLB-amp (LB broth with 100 μg/ml ampicillin) soft agar (0.5% agar) cooledto 45° C., and immediately overlaid upon an LB-amp agar plate. Theplates were inverted and incubated overnight at 37° C. The titer wasdetermined by the number of ampicillin-resistant colonies that haveformed at each dilution.

[0047] EGFR-specific scFvs were recovered from the phage library byabsorbing loll to 10¹² cfu in 4 ml PBS-T to the EGFR-coated immunotubeswith rocking for 2 hours at RT. The immunotubes were washed 20 timeswith PBS-T followed by 20 times with PBS. The EGFR-specific virus waseluted in 1 ml 100 mM triethylamine (Sigma) for 5 min at roomtemperature, then immediately neutralized with 1 ml 1 M Tris-HCl, pH 7.4and stored on ice until ready to infect XL1-Blue cells.

[0048] To amplify the EGFR-specific scFv bacteriophage, XL1-Blue cellswere grown in 20 ml LB-tet broth until an OD₆₀₀ of 0.4. Theneutralized-eluted phage was added to the culture, allowed to adsorbundisturbed for 15 min at 37° C., followed by shaking (240 rpm) for 45min. An aliquot was remove (200 μl) to determine the cfu titer, bymaking 10-fold dilutions of the 200 μl aliquot in SOB-GA broth (up to10⁻⁴), then 100 μl from each dilution was spread onto an SOB-GA agarplate (100 mm²) and incubated overnight at 37° C. The cfu titer wasdetermined by counting the number of ampicillin resistant colonies. Theremaining cells were separated from the broth by centrifugation at2000×g for 5 min at RT. The cell pellet was suspended in 1000 μl SOB-GAmedium (20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 50 mM MgSO₄, 100mM glucose, 100 μg/ml ampicillin), then spread onto 3 SOB-GA agar plates(150 mm²). After the plates dried, they were inverted and incubated at37° C. for 18-24 h. The colonies grown on the 150 mm² plates wereremoved by scraping the bacteria into 10 ml SOB-GA broth per plate. Thebacteria were pooled, then used to inoculate a 250 ml 2xYT-GA brothculture to an OD₆₀₀ of 0.025. Glycerol was added to a concentration of20% to the remaining bacteria and stored at −80° C. To increase thespecificity, the process for isolating EGFR-specific scFvs was repeated2 additional times.

[0049] Putative anti-EGFR-specific scFv clones were isolated from the2nd round and 3rd round of screening. All clones were stored at −80° C.as a 20% glycerol stock of an overnight broth culture grown in 2xYT-GAbroth.

[0050] Small Scale Phage Rescue

[0051] The scFv bacterial clones were used to inoculate 0.2 ml 2xYT-GAmedium and grown overnight at 37° C. Ten μl of the overnight culture wastransferred to 1 ml 2xYT-GA medium and incubated with shaking (300 rpm)at 37° C. for 3 hours. M13K07 helper phage (10¹⁰ cfu) was added to eachculture, mixed gently and set undisturbed for 15 min at 37° C., thenshaked for 45 min at 300 rpm. The cells were separated by centrifugationat 1000×g for 5 min at room temperature and the supernatant removed,then 1 ml 2xYT-AK medium was added to the cell pellet and incubated withshaking for 7 hours. The cells were removed by centrifugation and thesupernatant collected. The supernatant was stored at 4° C. for u p to 3days.

[0052] Screening Clones by Phage ELISA

[0053] The 96-well MaxiSorb immunoplates (Nunc, Rochester, N.Y.) werecoated with 1 μg/ml affinity purified EGFR antigen (Sigma) in 100 mMsodium carbonate, pH 9.6 buffer. The antigen was allowed to adsorbovernight at 4° C., then the antigen was removed and the wells werewashed 3 times with PBS-T. The wells were blocked with 2% skim milk-PBSfor 2 hours at room temperature. The wells were washed 3 times withPBS-T, then 100 μl of rescued phage was added per well and incubated atroom temperature for 2 hours. The wells were washed 3 times with PBS-T,then 100 μl of a {fraction (1/1000)} dilution of anti-M13-HRP(Stratagene, LaJolla, Calif.) was added to each well and incubated for 1h at room temperature. The wells were washed 5 times with PBS-T, then200 μl of the TMB enzyme substrate (Sigma) was added per well. The ELISAplates were incubated at room temperature for 30 minutes, then read at650 nM. Wells equal to and above OD₆₅₀=0.1 were considered positive andbelow OD₆₅₀=0.1 were considered negative.

EXAMPLE 2

[0054] Sequencing and Analysis of scFv Clones

[0055] After 3 rounds of phage panning, individual clones wereidentified by ELISA as described above. Plasmid DNA was isolated andsequenced according to standard manufacturer's protocol for the ABI DNAsequencer (UAB Automated DNA Sequencing Core Facility). Plasmid DNA wassequenced both directions initially using pelB and gene III primers.Internal sequencing primers were determined from the initial sequencedata and synthesized by Operon (Alameda, Calif.). After completion ofthe scFv sequence, the data was analyzed using SeqWeb software (GeneticsComputer Group, Madison, Wis.) for alignment ofcomplementary-determining regions (CDRs) with known variable-chainsequence data.

[0056] Two clones, pSEX81-6 and pSEX81-63, have been sequenced and theirputative amino acid sequences are shown in FIG. 2. The clones are in theorder, variable heavy chain (V_(H))-linker-variable light chain (V_(L)),with both clones containing a lambda V_(L) chain.

[0057] When comparing the two clones, there is a 48% amino acid identityin the V_(H) chain and an 87% amino acid identity in the V_(L) chain.The hypervariable or complementarity-determining regions (as defined inref. 16) are located at the tips of the Fabs in a 3-dimensionalstructure and have been shown to be primarily involved with antigenbinding (17). The CDR1-L region is 100% identical between the twoclones, whereas the other CDRs vary from 2 amino acid differences inCDR3-L, CDR2-L and CDR1-H to 10 and 12 amino acid differences in CDR3-Hand CDR2-H, respectively. With the high variability between the CDRs ofthese two clones, each clone may bind to a different antigenic site onthe EGFR.

EXAMPLE 3

[0058] Targeting the scFv to a Cellular Compartment and Expression ofSecretory scFv

[0059] In eukaryotic cells, scFvs can be targeted to specificsubcellular compartments by engineering the nucleotide sequence toexpress a protein with the appropriate signal sequences. In this way thescFvs can be modified to be directed to a subcellular compartment wherethe antibody might prove to be most effective. Recently, Lotti et al.showed that the C-terminal sequence KKXX from the adenovirus E19 proteinwould enhance the localization of the protein to the cis-golgi complexwith some retention in the ER (18). To direct the scFv to the cytoplasm,the hydrophobic amino acid core of the immunoglobulin secretory signalsequence was removed (19). The addition of a nuclear localization signalfrom the large T-antigen of SV40 virus, PKKKRKV (SEQ ID No. 3), to theN-terminal end can target the scFv to the nucleus (20).

[0060] To target mitochondria, the N-terminal presequence of the subunitVIII of human cytochrome c oxidase was added to the N-terminal end ofthe scFv (21). Other investigators have directed scFvs to the lumen ofthe endoplasmic reticulum by including the endoplasmic reticulumretention signal (SEKDEL, SEQ ID No. 4) at the C-terminus of thepolypeptide (22-25) scFvs can also be directed to the secretory pathwayby the addition of an immunoglobulin signal peptide on the N-terminalend (23-27).

[0061] In order to express the secretory scFvs in eukaryotic cells, theinsert encoding the scFvs must be cloned downstream of an IgK secretoryleader sequence in a eukaryotic expression vector. To this end, theeukaryotic expression vector, pSecTag (Invitrogen) was modified at themultiple cloning site to accept the restriction enzyme sites (Bpul102Iand NotI), in the correct orientation and in the proper reading framebetween the leader sequence and the myc (mAB 9E10 epitope) and (His)₆tags. Also, the eukaryotic antibiotic resistance gene was changed fromzeomycin to neomycin, thus the plasmid is named pSecTag/Bpu/neo (FIG.3).

[0062] Standard cloning techniques were employed to clone the scFv intopSecTag/Bpu/neo. Briefly, 20 ml LB-amp medium were inoculated withXL1-Blue cells expressing the pSEX81-scFv. After an overnight incubationat 37° C., the bacteria were recovered by centrifugation at 4000×g for 5min. The plasmid DNA were isolated using a Wizard DNA Purification kit(Promega, Madison, Wis.). The pSEX81-scFv plasmid DNA were digested withthe restriction enzymes, Bpu1101I and NotI, and separated by agarose gelelectrophoresis. The scFv fragment were recovered from the agarose gelusing AgarACE Enzyme (Promega). T4 DNA ligase were used to ligate thescFv fragment into the pSecTag/Bpu/neo plasmid which was digested withBpu1101I and NotI and agarose gel purified. The ligated DNA was use totransform E. coli Top 10F′ competent cells (Invitrogen) and plated ontoLB-A (LB medium with 100 μg/ml ampicillin) agar plates. After anovernight incubation at 37° C., individual colonies were selected andused to inoculate 5 ml LB-A broth cultures. Plasmid DNA was recoveredusing the Wizard DNA Purification kit and analyzed forpSecTag/Bpu/neo-scFv by PCR, amplifying the scFv clone between a T7promoter primer and a myc tag primer. No insert produced a 248 bp PCRproduct and a positive clone produced a PCR product between 800-1200 bp.

[0063] The positive clones were amplified in their bacterial host andthe plasmid DNA was recovered using the Wizard PureFection Plasmid DNAPurification System (Promega). The low EGFR expressing human glioma cellline U87MG was transiently transfected with insert positive plasmid DNAor vector alone using the Lipofectin Reagent (Life Technologies).Forty-eight hours after transfection, culture medium was collected andanalyzed for secretory scFv to EGFR Stable transfects were isolated byselecting for antibiotic G-418 resistance with the scFv clones(U87MG-scFv) and vector clones (U87MG-pSecTag/Bpu/neo). The stabletransfects were subcultured in 96-well plates at a density, of less than1 cell per well. The culture medium from the confluent wells werescreened by ELISA testing for secreted anti-EGFR-scFv. The positivesubclones were subcultured in 96-well plates at a density of less than 1cell per well and the wells grown to confluent monolayers were screenedfor secreting anti-EGFR-scFv by ELISA. The positive subclones wereexpanded for further analysis.

EXAMPLE 4

[0064] Screening for Anti-EGFR-scFv by Immunoblot and ELISA

[0065] One of the stably transfected human glioma sublines,U87MG.6.34.A8 (referred to as clone p6.34) was tested for its ability tosecrete a functional, anti-EGFR scFv. The anti-EGFR-scFvs wereimmunoprecipitated from cell lysates and culture medium. Briefly, thecells were placed on ice, washed three times with ice-cold PBS, lysed inice-cold lysis buffer (containing 0.025 M Tris-HCl, pH 7.5; 0.25 M NaCl,0.005 M EDTA, 1% v/v NP-40, 0.001 M phenylmethylsulfonylfluoride, 15μg/ml aprotinin, 10 μg/ml leupeptin, 0.001 M Na-orthovanadate, 0.05 MNa-fluoride and 0.03 M Na-pyrophosphate) then clarified bycentrifugation at 15,000×g for 20 min at 4° C. Protein concentrationswere determined using a BCA protein assay kit (Pierce). Equal amounts ofprotein were immunoprecipitated with mouse anti-myc antibody (9E10epitope, Stratagene) using Protein A/G beads (Pierce). Forimmunoprecipitation of secretory scFvs from the culture medium,four-day-old cell culture supernatants were collected from the stablytransfected cells. Equal volumes (1 ml) were immunoprecipitated with ratanti-tubulin antibody (Serotec Inc., Raleigh, N.C.) using Protein A/Gbeads.

[0066] Screening for the expression and secretion of the scFvs was byimmunoblot, whereby the immunoprecipitated proteins were denatured byboiling in sample buffer (0.125 M Tris-HCl, pH 6.8; 10% glycerol, 1%SDS, 0.7 M β-mercaptoethanol and 0.25% bromophenol blue) for 3 min andseparated by SDS-PAGE then transferred to Immobilon-P membrane(Millipore Corp., Bedford, Mass.). Immunoblots were blocked in 10%milk-TBS-T (Tris-buffered saline with 0.05% Tween-20) for 1 hour at roomtemperature. Primary antibody, mouse anti-myc (Stratagene) was incubatedin 2% milk-TBS-T overnight at 4° C. Blots were washed three times inTBS-T followed by incubation with HRP (horseradish peroxidase) labeledsecondary antibody, anti-mouse Ig-horseradish peroxidase antibody(Sigma) at room temperature for 1 hour. The blots were washed threetimes with TBS-T and once with TBS. The blots were developed bychemiluminescence (Amersham Pharmacia Biotech, Piscataway, N.J.).

[0067] Results show that scFv was immunoprecipitated from the celllysate (FIG. 4A) and the culture medium. (FIG. 4B) of clone p6.34 butnot from the control cell line pSECTAG that was stably transfected withthe parent vector pSECTAG/Bpu/Neo. These data indicated that scFv wastranslated and processed into the secretory pathway. However, these datadid not indicate whether clone p6.34 scFv binds to the EGFR antigen.

[0068] Therefore, culture medium from stably transfected cells wastested in an ELISA for the expression of secretory scFv against EGFR.The culture medium was clarified by low speed centrifugation (1000×g for5 min at 4° C.) to remove any cells, then clarified at high speedcentrifugation (10,000×g for 15 min at 4° C.) to remove any debris. Cellculture medium (100 μl) was added to each well and incubated for 2 h at37° C. in a CO₂ incubator. The wells were washed with PBS-Tween-20(PBS-T). Secondary antibody, anti-myc (mAb 9E10, Invitrogen) at a 1:1000dilution in PBS-T was added to each well and incubated for 1 hour at 22°C. After washing the wells, a tertiary antibody, anti-mouse-HRP (Sigma)at 1:2000 dilution in PBS-T was added to each well, then incubated for 1h at 22° C. The wells were washed and developed with o-phenylenediamine(OPD, Sigma) and read at 450 nm. The results shown in FIG. 4C indicatethat clone p6.34 secretes scFv that binds to the purified EGFR antigen.

EXAMPLE 5

[0069] Binding of Anti-EGFR-scFv to the Extracellular Domain of EGFR

[0070] The ELISA data shown above indicates that clone p6.34 scFv bindsto denatured EGFR, however it does not provide any information as towhich part of the receptor was recognized by the scFv. The EGFR hasthree major domains, intracellular, transmembrane and extracellular, anyof which may serve as the binding site for clone p6.34. To examinewhether the scFv binds to the extracellular portion of the receptor, aFACS analysis was used for this determination. For this assay, culturemedium collected from U87MG.pSECTAG or U87MG.pSECTAG.6.34.A8 was allowedto interact with the cell surface of 3 different human glioma sublines;U87MG, U87MG.wtEGFR and U87MG.AEGFR (provided by Dr. H-J. Su Huang,UCSD).

[0071] The U87MG is the parent cell line into which the scFv clones werestably transfected as well as stably transfected with wild-type EGFR(U87MG.wtEGFR) or the truncated EGFR, EGFRvIII (U87MG.ΔEGFR) (28-29).U87MG has a very low number of EGFR, which is one reason why cellproliferation of the stably transfected cell line, clone p6.34, does notappear to be affected by the anti-EGFR scFv (data not shown). TheU87MG.wtEGFR subline overexpresses a large number of EGFR/cell(estimated at >3×10⁶). The U87MG.ΔEGFR expresses the 135 kdal truncatedEGFR which is constitutively phosphorylated (29).

[0072] The FACS results shown in FIG. 5 indicates that the secretoryscFv p6.34 binds to the extracellular domain of U87MG.wtEGFR andU87MG.ΔEGFR. The parent cell line, U87MG, does not have a significantnumber of receptors on its cell surface which results in no detectablescFv p6.34 binding. The data indicates that clone p6.34 produced asecretory scFv which bound to the cell surface of cells whichoverexpress EGFR and truncated EGFR (EGFRvIII). Since clone p6.34 boundto a common antigenic site on both prominent forms of cancer-relatedEGFRs, this scFv might block the surface expression of each receptorwhen presented in the proper subcellular compartment.

EXAMPLE 6

[0073] Radiolabelled Anti-EGFR-scFv for Early Detection of Breast Cancer

[0074] Many tumor-specific antigens have been identified as targets forimaging breast cancer. HER-2/neu is overexpressed on 25%-30% of breastcancer cells. The epidermal growth factor receptor, a receptor in thesame family as HER-2/neu, has been found to be overexpressed in a highpercentage of human carcinomas. A compilation of the literatureestimates that approximately 30%-35% of the breast carcinomas haveincreased levels of epidermal growth factor receptor protein and theincrease of epidermal growth factor receptor expression correlates withthe loss of estrogen receptor and a poor prognosis.

[0075] With the increasing data on the relationship between theoverexpression of the epidermal growth factor receptor and poorprognosis, this receptor has become a target for breast cancer imaging.Radiolabelled monoclonal antibodies (mAbs) have been used for imagingepidermal growth factor receptor-overexpressing breast tumors. However,due to the heterologous vascular structure around the tumor and themolecular size of the antibodies, the monoclonal antibodies penetratethe tumor poorly and are unevenly distributed around the tumor makingimaging more difficult.

[0076] In order to improve tumor imaging, intact monoclonal antibodieshave been reduced to antibody fragments or single-chain antibodies. Theshort plasma half-life for the scFv becomes an advantage for tumorimaging because tumor-to-blood ratios are higher than intact monoclonalantibodies and the rapidly eliminated scFv does not accumulate inextravascular spaces and non-target organs. Isotopes used for imagingtumors include ¹¹¹In, ⁶⁴Cu, ¹³¹I, ⁹⁹Y, and ⁹⁹ mTc. Technetium-99m isused in the following protocol because the techniques are available forboth direct and indirect scFv labelling and ⁹⁹ mTc is a low cost,readily available isotope with purely photon radiation, which is widelyused in clinical imaging. The 6 h half-life of ⁹⁹ mTc is an excellentcomplement to the scFv which has a rapid clearance from the circulationsystem allowing for high-contrast imaging.

[0077] The radiolabelled scFv of the present invention will b e testedfor any loss of ability to bind to the epidermal growth factor receptordue to the radiolabelling process. Then, the radiolabelled scFv can beuse in mouse xenograft models to determine its ability to bind to largeor small xenografts of high, moderate or low epidermal growth factorreceptor overexpressing breast cancer cells detected by a gamma camerawith computer-enhanced imaging.

[0078] Although tissue biodistribution is currently a standard method toassess pharmacokinetics in mice, it suffers from several drawbacks.First, the concentrations of the radiolabelled peptides at each timepoint are measured from different mice and data are pooled for eachseparately evaluated group. Second, the number of time points to besampled are often limited to 3-5 points, consequently, the fast phase oftissue uptake is ignored. Because of the small size of theanti-epidermal growth factor receptor-scFv, the uptake and clearance areexpected to be fast in this study. Thus, the pharmacokinetics of the ⁹⁹mTc-scFv will be determined b y dynamic imaging using a pinhole gammacamera, and the results are useful for the development of non-invasiveimaging method for early detection of breast cancer.

[0079] Breast Cancer Cell Lines

[0080] The following cell lines are purchased from ATCC (Manassas, Va.):MDA-MB-468, MDA-MB-231 and MCF-7. These cell lines are reported tooverexpress EGFR at levels ranging from low (10,000 receptors/cell,MCF-7), medium (100,000 receptors/cell, MDA-MB-231) and high (1,000,000receptors/cell, MDA-MB-468).

[0081] Radiolabelling of the anti-EGFR scFv

[0082] The indirect radiolabelling procedure, with the trihydroxamatbifunctional chelating agent trisuccin (42), is used for ^(99m)Tclabelling of the scFv. The protocol is in two steps consisting ofconjugation of trisuccin to the scFv (43), followed by theradiolabelling of the conjugate.

[0083] For trisuccin conjugation, the 50 μl solution of the scFv in DPBSis added to 250 μL of a 50 mM PBS buffer at -pH 8.1. The resultingsolution is stirred at 0° C. and the solution of OHA-NHS (5.7 g, 23.5nmol) in 50 μl of DMF is added. After 90 min at 0° C., the reaction isquenched by addition of 20 μl of 2 M glycine in the PBS buffer and thereaction mixture is purified by dialysis against 100 mM acetate buffer,pH 5.5. To this mixture, a solution of trisuccin hydrazide. (43) (0.12mol -1.2 mol) in water (5 μl-20 μl) is added. The reaction mixture isstirred at room temperature for 90 min a t which time 20 μl of NaCNBH₃solution in water is added to a final concentration of 100 mM inNaCNBH₃. The resulting mixture is stirred gently at room temperature for18 h followed b y purification of the conjugate by size-exclusion HPLC(SEC-HPLC) and eluted with DPBS.

[0084] For ^(99m)Tc-labelling, the procedure reported previously is used(42, 43). Briefly, the ^(99m)Tc, eluted from a ⁹⁹Mo/^(99m)Tc generator(Syncor, Birmingham, Ala.) is reduced with a solution of SnCl₂ inhydroxyisobutyric acid at room temperature. This solution is added tothe trisuccin- scFv and incubated at 35° C. for 30 min. The labelledprotein is purified by SEC-HPLC.

[0085] Alternately for direct ^(99m)Tc labelling, cysteine molecules onthe purified scFv is radiolabeled with ^(99m)Tc by previously publishedmethods using a ^(99m)Tc-D-glucarate transfer method (44, 45). Moleculartechniques is used to add a cysteine molecule near the COOH-terminal endof the scFv which will be available for this site directed labellingmethod. The radiolabelled protein is purified by SEC-HPLC.

[0086] Testing the Binding Ability of the Radiolabelled scFv

[0087] Whole cells or cell membranes from the three breast cancer celllines were used in a standard competitive radioimmunoassay to determineif the radiolabelled scFv retains its ability to bind to the EGFR. Theradiolabelled scFv used in competition with unlabelled scFv and assayswere counted in a gamma counter.

[0088] Human Breast Cancer Xenograft Models

[0089] Initially, 3 groups of 3 female athymic nude mice, 6 to 8 weeksold, obtained from the National Cancer Institute Frederick ResearchLaboratory (Frederick, Md.) are injected subcutaneously in the one flankwith 2×10⁷ human breast cancer cells. Each group is injected with one ofthe three breast cancer cell lines, MDA-MB-468, MDA-MB-231 and MCF-7.When the tumors are approximately 10 mm in diameter, the opposite flankare injected with the same cell line. When the second xenograft isapproximately 1 mm in diameter, the mice are injected with the^(99m)Tc-scFv and imaged. The xenograft model are repeated 2 additionaltimes and modified if necessary to produce consistent, reproducibleresults.

[0090] In Vivo Detection of Breast Cancer Tumors

[0091] Planar imaging of mice is performed using Picker Axis gammacamera equipped with a pinhole collimator with 2-mm or 4-mm aperture.The images are acquired with a matrix of 256×256 pixels and a zoomfactor of 2. Studies are performed using a 20% energy window, centeredon 140 keV photo-peak of ^(99m)Tc. Mice are imaged immediately afterbeing anesthetized. Previous imaging studies suggest that most micecould be imaged for 45-60 minutes before they recover from theanesthesia. Each mouse receives 50 μCi ^(99m)Tc-scFv IV. Mice are imagedin a prone position as xenografts are planted subcutaneously in bothventral flanks. A distance of 7.5 cm from pinhole to mice is used forimaging a group of 3 mice simultaneously in one field of view. Dynamicimage is performed at 0-1 h, 60 frames of images are acquired at 1min/frame rate to obtain fast phase for scFv uptake in normal tissue andthe tumor. Static images are acquired at 2 h, 4 h, 6 h, respectively.Acquisition time of each image is 20-30 min, which is comparable to thatof patient imaging using a gamma camera.

[0092] These images are used to determine the optimal imaging time andbest tumor to non-tumor image contrast that can be used for futurepatient imaging. This imaging computerized kinetic model allows one toobtain pharmacokinetics information in same animals and provide directevidence for the potential of using ^(99m)Tc-human anti-EGFR-scFv forearly detection of breast cancer in patients.

[0093] The following references were cited herein:

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[0108] 15. Koch and Dübel, 2000. Generation of antibody libraries fromhuman donors. In: Antibody Engineering. Eds: R. Konterman and S. Dubel.Springer Verlag, Heidelberg/NewYork.

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[0123] 30. Winter et al., Annual Review of Immunology. 12: 433-55, 1994.

[0124] 31. Marks, et al., Journal of Molecular Biology. 222: 581-97,1991.

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[0127] 34. Barnes et al., Clinical Cancer Research. 2: 1089-95, 1996.

[0128] 35. Stackhouse et al., Intl. J of Radiation Oncology, Biology,Physics. 42: 817-22, 1998.

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[0134] 41. Chowdhury et al., PNAS. 95: 669-74, 1998.

[0135] 42. Safavy et al., Bioconjugate Chemistry, 4: 194-8, 1993.

[0136] 43. Safavy et al., Bioconjugate Chemistry, 10: 18-23, 1999.

[0137] 44. Verhaar et al., Journal of Nuclear Medicine, 37: 868-72,1996.

[0138] 45. Pak et al., International Journal of Radiation Applications &Instrumentation—Part B, Nuclear Medicine & Biology, 19: 669-77, 1992.

[0139] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0140] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1 2 1 268 PRT artificial sequence amino acid sequence of anti-EGFR scFVclone pSEX81-6 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln ProGly 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser20 25 30 Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 3540 45 Glu Tyr Val Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr 50 5560 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 80 85 90 ThrAla Val Tyr Tyr Cys Val Lys Asp Val Gly Gly Ser Ser Trp 95 100 105 TyrTrp Ala Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 110 115 120 ThrVal Ser Ser Gly Ser Ala Ser Ala Pro Lys Leu Glu Glu Gly 125 130 135 GluPhe Ser Glu Ala Arg Val Gln Ser Val Leu Thr Gln Pro Pro 140 145 150 SerLeu Ser Val Ser Pro Gly Gln Thr Ala Ser Ile Thr Cys Ser 155 160 165 GlyAsp Lys Leu Gly Asp Lys Tyr Ala Ser Trp Tyr Gln Gln Lys 170 175 180 ProGly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Arg Lys Arg 185 190 195 ProSer Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn 200 205 210 ThrAla Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala 215 220 225 AspTyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Pro Tyr Val Phe 230 235 240 GlyThr Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys Ala Asn 245 250 255 ProThr Val Thr Leu Phe Pro Pro Ser Ser Ala Ala Ala 260 265 2 270 PRTartificial sequence amino acid sequence of anti-EGFR scFV clonepSEX81-63 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly5 10 15 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser 2025 30 Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 35 4045 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr 50 55 60Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser 65 70 75 ThrSer Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 80 85 90 Thr AlaVal Tyr Tyr Cys Ala Arg Asp Pro Asp Tyr Tyr Gly Ser 95 100 105 Gly SerTyr Tyr Pro Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr 110 115 120 Leu ValThr Val Ser Ser Gly Ser Ala Ser Ala Pro Lys Leu Glu 125 130 135 Glu GlyGlu Phe Ser Glu Ala Arg Val Gln Ser Ala Leu Thr Gln 140 145 150 Pro ProSer Val Ser Val Ser Pro Gly Gln Thr Ala Ser Ile Thr 155 160 165 Cys SerGly Asp Lys Leu Gly Asp Lys Tyr Ala Ser Trp Tyr Gln 170 175 180 Leu LysPro Ala Gln Ser Pro Val Trp Val Ile Tyr Gln Asp Thr 185 190 195 Lys ArgSer Ser Gly Ile Pro Glu Arg Ile Ser Gly Ser Asn Ser 200 205 210 Gly AsnThr Ser Thr Leu Thr Ile Thr Gly Thr Gln Ala Met Asp 215 220 225 Glu AlaAsp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val 230 235 240 Val PheGly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 245 250 255 Ala AsnPro Ser Val Thr Leu Phe Pro Pro Ser Ser Ala Ala Ala 260 265 270

What is claimed is:
 1. A human anti-epidermal growth factor receptorsingle-chain antibody having the sequence of SEQ ID No. 1 orbiologically functional fragment thereof.
 2. The anti-epidermal growthfactor receptor single-chain antibody of claim 1, wherein said antibodybinds to the extracellular domain of epidermal growth factor receptor.3. A human anti-epidermal growth factor receptor single-chain antibodyhaving the sequence of SEQ ID No. 2 or biologically functional fragmentthereof.
 4. The anti-epidermal growth factor receptor single-chainantibody of claim 3, wherein said antibody binds to the epidermal growthfactor receptor.
 5. An isolated DNA encoding the anti-epidermal growthfactor receptor single-chain antibody of claim
 1. 6. An isolated DNAencoding the anti-epidermal growth factor receptor single-chain antibodyof claim
 3. 7. An expression vector comprising the anti-epidermal growthfactor receptor single-chain antibody of claim
 1. 8. An expressionvector comprising the anti-epidermal growth factor receptor single-chainantibody of claim
 3. 9. A pharmaceutical composition comprising theanti-epidermal growth factor receptor single-chain antibody of claim 1and a pharmaceutically acceptable carrier.
 10. The pharmaceuticalcomposition of claim 9, further comprising a therapeutic agent.
 11. Thepharmaceutical composition of claim 10, wherein said therapeutic agentis selected from the group consisting of a toxin, a chemotherapeuticagent, a radioisotope and gene therapy vector.
 12. The pharmaceuticalcomposition of claim 9, further comprising a diagnostic agent.
 13. Thepharmaceutical composition of claim 12, wherein said diagnostic agent isselected from the group consisting of radioisotopes and transitionmetals.
 14. A pharmaceutical composition comprising the anti-epidermalgrowth factor receptor single-chain antibody of claim 3 and apharmaceutically acceptable carrier.
 15. The pharmaceutical compositionof claim 14, further comprising a therapeutic agent.
 16. Thepharmaceutical composition of claim 15, wherein said therapeutic agentis selected from the group consisting of a toxin, a chemotherapeuticagent, a radioisotope and gene therapy vector.
 17. The pharmaceuticalcomposition of claim 14, further comprising a diagnostic agent.
 18. Thepharmaceutical composition of claim 17, wherein said diagnostic agent isselected from the group consisting of radioisotopes and transitionmetals.
 19. A method of treating a tumor, comprising administering to apatient in need of such treatment an effective amount of ananti-epidermal growth factor receptor single-chain antibody of claim 1.20. A method of treating a tumor, comprising administering to a patientin need of such treatment an effective amount of an anti-epidermalgrowth factor receptor single-chain antibody of claim
 3. 21. Theanti-epidermal growth factor receptor single-chain antibody of claim 1,wherein said antibody is radiolabeled.
 22. The anti-epidermal growthfactor receptor single-chain antibody of claim 3, wherein said antibodyis radiolabeled.
 23. A method for the diagnostic location and assessmentof tumor growth, comprising administering to a patient in need of suchdiagnostic treatment an effective amount of an anti-epidermal growthfactor receptor single-chain antibody of claim
 21. 24. A method for thediagnostic location and assessment of tumor growth, comprisingadministering to a patient in need of such diagnostic treatment aneffective amount of an anti-epidermal growth factor receptorsingle-chain antibody of claim 22.